PCR-based screening: A Solid 3Rs Choice During Quarantine

Some best practices when using sentinel-free soiled bedding sampling as an alternative method for finding rodent pathogens during quarantine

As new complex genetic lines of rodents are produced, interest in their use among collaborators means they don’t stay in one place for very long. The facilities that create them will breed them and provide them to other institutions, often globally, where they support a variety of related research studies in pursuit of therapies. How, then, can the facilities shipping and receiving research animals ensure that the animals in their care don’t introduce unwanted pathogens that can contaminate the entire research rodent population? How can they ensure that animal health and the integrity of their research are preserved?

Traditionally, laboratories monitor the health of quarantined animals by probing soiled bedding sentinels, a labor-intensive system that also requires fairly large numbers of animals. More recently, however, a growing number of laboratories have turned to 3Rs-friendly PCR-based direct screening methods, and more recently PathogenBinder®, which eliminates direct sampling and handling of mice. With regulatory authorities placing an increased emphasis on non-animal alternatives in drug development and chemical testing, the number of laboratories using PCR-based methods to detect pathogens during quarantine is expected to continue growing. Which begs the question: Are there any rules that specifically address how to use these non-animal methods during quarantine? We’re glad you asked.

Giving it a “Fair Shake”

PathogenBinder^® relies on shaking or agitation of a high-binding collection media to collect pathogen-associated dust in soiled bedding. For sentinel-free surveillance methods, such as PCR-based PathogenBinder^®, which captures any pathogens to which animals are exposed during quarantine, we aim to develop a protocol that maximizes infectious agent detection. Getting the protocol right the first time is crucial for the process adoption in the community, says Ken Henderson, PhD, Charles River’s Senior Director of Laboratory Scientific Diagnostic Services and one of the longtime pioneers in the development of non-animal alternatives to dirty bedding sentinels. If we fail to deliver a working protocol, we could compromise research models, not to mention taking a step backwards in integrating  3Rs methods.

It was essential to collect pilot study and field study data to verify what the sampling protocol needed to include in order to reliably build up pathogen-associated material in the bedding over time for adequate detection. General protocol requirements were hypothesized, but confirmation of the quarantine protocol was solidified through a pilot and field study conducted by Charles River and an academic collaborator, Rutgers University. The investigations compared PathogenBinder^® with direct sampling of quarantined rodents, considered the gold standard for quarantine methods today. The findings, published earlier this year in the Journal of the American Association for Laboratory Science, showed the “hands-off” PathogenBinder^® approach to be a reliable and effective alternative, at least equivalent to direct sampling methods that required rodent handling.

Although the studies utilized two shake sampling events, one sampling of the transport bedding upon arrival, and a second event after the transport bedding spent an additional week with the rodents, it was determined that continued agent shedding over the one-week period was critical. Along with additional investigations within Charles River, it was determined that if transport bedding could be maintained with the quarantined rodents for an additional week, agent-associated particles containing their DNA or RNA were detected without the need to shake twice.   Therefore, only a single shaking event at the end of a one-week holding period was needed, which reduced the hands-on labor. The data is now helping to shape protocols for laboratories that wish to use sentinel-free surveillance systems during quarantine. 

Henderson, whose research has informed best practices for PCR-based exhaust air dust testing and, most recently, PathogenBinder^®, said the investigation into a PathogenBinder^® quarantine protocol was launched because a research animal facility indicated that it planned to use PathogenBinder^® as a clearance tool for moving mice from one building to another. “One of their buildings had pinworms, and the facility wanted to test the animals before shipping them over,” he said. “After we realized that some institutions were moving mice based on a single shake of the collection box, it forced us to ask the questions: How well does sentinel-free surveillance work in a quarantine situation, and can we find agents with a single shake?”  Our investigation with Rutgers and the subsequent studies suggested that a single shake and an extended shaking time provided data and confidence to release a standard PathogenBinder^® quarantine protocol for the industry.

Based on these research findings, Henderson said experts now recommend the following when using sentinel-free surveillance systems during quarantine:

• On the day the shipped mice arrive, soiled bedding from the shipping crate and any supplemental bedding required are maintained with the incoming rodents in quarantine housing.  
• After an additional week (6-8 days), the bedding from the quarantined cages is removed and placed in a collection box or a clean cage, then agitated for 30 seconds with high-particle binding contact media to sample the bedding.
• Sterile forceps are used to remove the contact media for placement in a media collection tube for shipment and PCR testing at a diagnostic laboratory.    

Using E-beam Technology to Further Protect Research Models

Employing new technologies often runs into roadblocks. “The first contact media released by diagnostic laboratories was not decontaminated, and through sterility testing,” said Henderson, “we could grow bacteria using standard broth media,” says Henderson. “For those vivaria using isolators to protect the health status of research mice or high-level biosecurity vivaria to protect delicate models, including immunodeficient mice, biosecurity measures, including the decontamination of husbandry materials, are required.”

Pathogenic or not, the unintended introduction of bacteria may impact rodent health or, at the very least, alter the existing microbiome, which could alter data.   The decontamination of contact media, namely through the use of electronic beam (E-Beam) radiation, provides the means to prevent the introduction of inapparent bacteria into the same work environment often shared with research mice. The process itself is not complicated, but the level of radiation must decontaminate the contact media without being too high as to damage or modify the media, and thus compromising the media’s ability to bind to particles. “We worked with our E-beam vendor to conduct studies to do just that,” said Henderson. “We assessed a range of E-Beam doses using sterility testing and pathogen detection studies to measure process success.” 

As part of the development process, map studies were conducted to demonstrate the minimum dose of E-Beam penetration into shipping containers. Gamma irradiation, commonly used for research animal feed and bedding, is less gentle than E-Beam but is required due to the density of these materials. “The novel use of E-beam decontamination for contact media checked all the boxes,” said Henderson.

In conclusion, Henderson stated that the use of PCR-based methods in detecting rodent pathogens during quarantine is a solid 3Rs improvement because it eliminates the need to collect samples from the animals. The hands-off approach with this method reduces animal stress as well as animal technician compassion fatigue, which are big wins for both the 3Rs philosophy and the well-being of our animal staff,” says Henderson.